The laboratory rat is the preferred rodent model in pre-clinical drug studies and encompasses the bulk of accumulated knowledge in drug development. Their larger size facilitates procedures otherwise difficult in mice, including studies using instrumentation, blood sampling, and surgeries. Although rats are more suitable than mice for pharmacological, toxicological, physiological, and many other biological assays, the ease of genetic engineering technologies has made the mouse the preeminent rodent model. However, the recent emergence of new and more precise gene targeting techniques for the rat has resulted in significant growth in the production of genetically-modified rats. In Phase I, we demonstrated the feasibility of combining custom site-specific Xanthomonas TAL Nuclease (XTNTM) [a.k.a. TALEN] technology with spermatogonial stem cells (SSCs) for rapid, cost-effective and precise genome engineering in the rat. Indeed, the Phase I studies enabled Transposagen to launch custom XTNTM and knockout rat production services in 2012. The proposed studies will address the unmet need for rat models for drug discovery applications. Creating knockout models for enzymes involved in drug metabolism, which are often arranged as closely linked members of multi-gene families, presents a new production challenge. In this proposal, we will develop strategies for creating large-scale deletions of multi-gene families combined with humanizing transgenic engineering to create novel and more predictive models for drug discovery. We will develop protocols to use XTNTMs to generate large-scale chromosomal deletions in SSCs that will eliminate whole gene families from the genome. To optimize the experimental approach, we will outline studies to delete the 2-gene MATE complex which is located within a 70-kb segment of Chromosome 10 and the 5-gene Cyp2d cluster located within a 60-kb segment of Chromosome 7. SSCs that harbor a 45- kb deletion that knocks out the entire Cyp2d complex will be transplanted into recipient males to create a Cyp2d null rat for CYP2D6 humanization studies. To create humanized rats, we will clone the wild type CYP2D6 gene (including its promoter) as well as four other common CYP2D6* variant genes into individual piggyBacTM (PB) vectors. The five PB-CYP2D6 constructs will be used to create five SSC lines that carry an average of one copy of the transgenic vector per cell, and founder animals will be generated from each SSC line. Each PB-CYP2D6 transgenic allele will be crossed into the Cyp2d cluster mutant background for phenotypic analysis. In the future, humanized animal models in which various combinations of CYP2D6* alleles would be of great value for testing the effect of drug candidates. The proposed Phase II studies will develop multiple protocols for genome engineering in the rat so that the most appropriate strategy can be executed to engineer the desired mutation. The success of this project will promote cost-effective technologies that can be used to create humanized models of choice in the appropriate null background for drug testing.